Antioxidant Activity Profiling by Spectrophotometric Methods of Aqueous Methanolic Extracts of Helichrysum Stoechas Subsp
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Chinese Journal of Natural Chinese Journal of Natural Medicines 2014, 12(6): 0415−0422 Medicines doi: 10.3724/SP.J.1009.2014.00415 Antioxidant activity profiling by spectrophotometric methods of aqueous methanolic extracts of Helichrysum stoechas subsp. ru- pestre and Phagnalon saxatile subsp. saxatile ,Tarik Mohammed Chaouche 1, Riadh Ksouri 2, Faten Medini 2 ,٭Farah Haddouchi 1 Fatima Zohra Sekkal 3, Abdelhafid Benmansour 1 1 Laboratory of Natural Products, Department of Biology, Faculty of Sciences, Abou Bekr Belkaïd University, B. P 119, Tlemcen, 13000, Algeria; 2 Laboratory of Plant Adaptation to Abiotic Stresses, Biotechnologic Center in Borj-Cedria Technopol (CBBC), B. P 901, 2050 Hammam-Lif, Tunisia; 3 Department of Biotechnology, Faculty of Natural Science and Life, Abdelel Hamid Ibn Badiss University, Mostaganem, 27000, Algeria Available online 20 June 2014 [ABSTRACT] AIM: The aqueous methanolic extracts of two plants from Algeria, Helichrysum stoechas subsp. rupestre and Phagnalon saxatile subsp. saxatile, were investigated for their antioxidant activity. METHOD: Total phenolics, flavonoids, and tannins were determined by spectrophotometric techniques. .In vitro antioxidant and radical scavenging profiling was determined by spectrophotometric methods, through: Total antioxidant capacity, and radical scavenging effects by the DPPH and ABTS methods, reducing and chelating power, and blanching inhibition of the β-carotene. RESULTS: All of the extracts showed interesting antioxidant and radical scavenging activity. The highest contents in phenolics, tannins, and the highest total antioxidant capacity as gallic acid equivalents of 97.5 ± 0.33 mg GAE/g DW was obtained for the flowers of H. stoechas subsp. rupestre extract in the phosphomolybdenum assay. An extract of the leafy stems of P. saxatile subsp. saxatile revealed the highest content of flavonoids, and the highest antioxidant activity by the radical scavenging and β-carotene assays when compared with standards. The best activity was by the scavenging radical DPPH with an IC50 value of 5.65 ± 0.10 µg·mL−1. CONCLUSION: The studied medicinal plants could provide scientific evidence for some traditional uses in the treatment of dis- eases related to the production of reactive oxygen species (ROS) and oxidative stress. [KEY WORDS] Helichrysum stoechas subsp. rupestre; Phagnalon saxatile subsp. saxatile; Phenolic compounds; Antioxidant activity [CLC Number] R965 [Document code] A [Article ID] 2095-6975(2014)06-0415-08 In order to prolong the storage stability of foods, and to re- Introduction duce damage to the human body, synthetic antioxidants, such When oxygen is supplied in excess, or its reduction is as butylated hydroxyanisole (BHA) and butylated hydroxyto- insufficient, reactive oxygen species (ROS) are generated. It luene (BHT), are commonly used. However, restrictions on is generally accepted that free radicals play an important role the use of these compounds are being imposed because of in the development of tissue damage and pathological events [1]. their carcinogenicity and some side effects [2]. Thus, evalua- tion of the antioxidant activity of naturally occurring sub- stances has been the focus of interest in recent years [3-4]. The [Received on] 20-Mar.-2013 antioxidant activity of plants, which contain a diverse group [*Corresponding author] Farah Haddouchi: Tel: 213-772735216, E-mail: [email protected] of phenolic compounds, is mainly due to their redox proper- These authors have no conflict of interest to declare. ties which make them act as reducing agents, hydrogen do- – 415 – Farah Haddouchi, et al. / Chin J Nat Med, 2014, 12(6): 415−422 nors, and singlet oxygen quenchers. They also may have a To each sample extract (100 µL) suitably diluted, Na2CO3 (2 metal chelating potential [5]. mL, 2%, W/V) was added, and the mixture shaken and The Helichrysum genus (Asteraceae) includes more than allowed to stand for 5 min. Folin–Ciocalteu reagent (100 500 species that are widespread around the world. A great µL, 1 mol·L−1) was added. After incubation for 30 min at number of biological activities are usually attributed to this room temperature in the dark, the absorbance versus a genus, such as anti-inflammatory, anti-allergic, antioxidant, prepared blank was read at 750 nm. Total phenolic content antimicrobial, cough relief, and treatment of colds and wounds was expressed as mg gallic acid equivalents per gram of [6]. The chemistry of the Helichrysum genus is complex, with dry weight (mg GAE/g DW) using a calibration curve for a wide variety of chemical classes, among which are flavono- gallic acid (0–400 µg·mL−1) ids, chalcones, phloroglucinol derivatives, essential oils, Total flavonoids α-pyrones, and diterpenes [7]. The genus Phagnalon (Astera- Measurement of flavonoid concentration in different ceae) is represented by about thirty species distributed aqueous methanolic extracts was based on the method de- worldwide, six of which are typical of the Mediterranean scribed by Dewanto et al [15]. An aliquot (250 µL) of the sam- [8] region . Different Phagnalon species are used in traditional ples was added to test tubes containing 5% NaNO2 solution medicine, and a variety of extracts have been examined [9]. (75 µL), and mixed for six min. Then, freshly prepared 10% Phytochemical investigations on Phagnalon species are AlCl3 solution (150 µL) was added, and after 5 min at room mainly devoted to the study of P. rupestre, and report the temperature, 1 mol·L−1 NaOH (0.5 mL) was added. The final presence of terpenoids [10], flavonoids [11], hydroquinone gly- volume was adjusted to 2.5 mL with distilled water and tho- cosides, and caffeoylquinic acid derivatives [12]. A survey of roughly mixed. The absorbance of the mixture was deter- the literature shows the presence in the aerial parts of P. sa x - mined at 510 nm against the same mixture without the sample atile of an essential oil [13] and flavonoids [10]. as a blank. Total flavonoid content was expressed as mg ca- However, scientific information on the antioxidant prop- techin per gram of dry weight (mg CE/g DW), through the erties of various plants, particularly those that are less widely calibration curve of (+)-catechin (0–400 μg·mL−1). used in the culinary arts and medicine is still rather scarce. Total condensed tannins There are any reports of the Helichrysum and Phagnalon The content of condensed tannins were determined by the species belonging to Algerian flora. The aim of the present procedure of Sun et al [16]. Fifty microliters of different work is to study the phenolic content and antioxidant activity aqueous methanolic extracts was mixed with 4% vanil- of aqueous methanolic extracts of Helichrysum stoechas lin-methanol solution (3 mL) and 1 mol·L−1 hydrochloric acid subsp. rupestre auct. and Phagnalon saxatile (L.) Cass. subsp. (1.5 mL). The mixture was allowed to stand for 15 min and saxatile, collected from Algeria. the absorbance was measured at 500 nm. The amount of total condensed tannins is expressed as mg catechin per gram of Material and Methods dry weight (mg CE/g DW). The calibration curve range of Preparation of plant extracts (+)-catechin was 0–400 μg·mL−1. The flowering aerial parts of the plants were collected in Determination of antioxidant activities March 2011, from two different regions of Tlemcen in the Total antioxidant capacity west of Algeria. H. stoechas subsp. rupestre was collected The assay is based on the reduction of Mo (VI) to Mo (V) from Honaine (35°10′35″ N, 1°39′18″ W) and P. saxatile subsp. by the extract and subsequent formation of a green phos- saxatile from Fellaoucen (35°02′06″ N, 1°36′21″ W). They phate/Mo (V) complex at acidic pH [17]. An aliquot (0.1 mL) were identified in the Laboratory of Natural Products, De- of aqueous methanolic extract was combined to 1 mL of rea- partment of Biology, University of Tlemcen, Algeria. Voucher gent solution (0.6 mol·L−1 sulfuric acid, 28 mmol·L−1 sodium specimens were deposited at the Herbarium of the Laboratory. phosphate and 4 mmol·L−1 ammonium molybdate). The tubes The plants were dried at room temperature for two weeks. were incubated in a thermal block at 95 °C for 90 min. After The flowers were separated from the leafy stems and the ex- the mixture had cooled to room temperature; the absorbance tracts were obtained separately by the magnetic stirring for 24 of each solution was measured at 695 nm against a blank. The h of the plant powder (2 g) with aqueous methanol 80 : 20 (25 antioxidant capacity was expressed as mg gallic acid equiva- mL). The aqueous methanolic extracts were evaporated at 45 lent per gram dry weight (mg GAE/g DW). The calibration °C under reduced pressure, re-dissolved in methanol at a curve range was 0–500 μg·mL−1. concentration of 10 mg·mL−1, and stored at 4 °C for further DPPH assay use. The antioxidant activity of the aqueous methanolic ex- Quantification of phenolic classes tracts of the plants was measured using the stable DPPH me- Total polyphenols thod based on the reduction of an alcoholic DPPH solution in The amounts of total phenolics in the aqueous methanolic the presence of a hydrogen donating antioxidant due to the extracts were determined with the Folin–Ciocalteu reagent formation of the non-radical form DPPH-H by the reaction [18]. using the method described by Vermerris and Nicholson [14]. The sample was diluted in pure extraction solvent at different – 416 – Farah Haddouchi, et al. / Chin J Nat Med, 2014, 12(6): 415−422 concentrations (0–3 mg·mL−1), then each diluted plant extract sessed as described by Zhao et al [21]. Different concentrations −5 −1 (50 μL) was added to a 6.34 × 10 mol·L DPPH methanolic of plant extracts were added to FeCl2 (4H2O) solution (50 µL, solution (1 950 μL).